Trout hepatomaFibrosis and lymphocytosis as suppressive mechanisms in the rainbow trout Salmo gairdneri.
код для вставкиСкачатьTrout Hepatoma : Fibrosis and Lymphocytosis as Suppressive Mechanisms in the Rainbow Trout (Sarimo gairdneri) ' J. H. WALES AND R. 0.SINNHUBER Department of Food Science and Technology, Oregon State University, Cornallis, Oregon 97331 ABSTRACT Fibrosis and lymphocytosis are common in aflatoxin-induced hepatoma in rainbow trout. It is suggested that these reactions may control the growth of tumors and in some cases destroy them. Nearly all of the hepatomas we have observed are fibrotic to some extent. The extent of the fibrosis seems to be age dependent and in advanced cases fibrosis appears to have disorganized the tumors to a degree which we believe is suppressive. The lymphocytosis shows a negative correlation with the size and age of the hepatoma, being found almost exclusively in the very "early" (small) hepatomas and preneoplastic nodules. It is suggested that the invasion of the tumors by lymphocytes is a host defense mechanism. of carcinogens and cocarcinogens have been tested under a wide variety of experimental conditions and in conjunction with many dietary modifications (Sinnhuber et al., '68). Large numbers of hepatomas can be produced by feeding any nutritionally balanced trout diet to which has been added aflatoxin B1 at levels within the range of 4 to 20 parts per billion (ppb). The carcinogen is added to the diet as soon as possible after feeding starts. Hepatomas may reach macroscopic size in four months although this is temperature-dependent and in water averaging 12" C large numbers of hepatomas are rarely obtained in less than six months. Livers containing hepatomas are routinely fixed in Bouin's fluid, embedded in paraffin and stained with hematoxylin and eosin. Masson's triple stain is occasionally used to emphasize the connective tissue in fibrotic tumors. In the course of this work, a very large number of hepatomas has been examined. Certain carcinogenic diets produce as many MATERIALS AND METHODS as 100 tumors in a single liver, ranging The brood stock of Mt. Shasta "strain" from microscopic size to more than 8 cm rainbow trout (Salmo gairdneri) main- in diameter. Unfortunately the observatained in our laboratory has been found to tions of fibrosis and lymphocytosis are diffibe more sensitive to aflatoxin-induced cult to treat statistically. Only a rough estihepatoma than any other animal tested. Received July 17, '72. Accepted Oct. 3, '72. 1This work was supported in part by U.S.P.H.S. Approximately 200 diet trials have been grants ES-00263 and ES-00256.Technical paper 3220, run in our laboratory since 1964. A number Oregon Agricultural Experiment Station. Infiltration of hepatomas in the rainbow trout by ductal tissue components (fibrosis) and by lymphocytes has been described by a number of workers (Haddow and Blake, '33; Cudkowicz and Scolari, '55; Nigrelli and Jakowska, '61; Wood and Larson, '61; Rucker et al., '61; Hueper and Payne, '61; Ghittino and Ceretto, '62; Ashley and Halver, '63; Scarpelli et al., '63; Ashley, '69; Wales, '70; Ashley, '70). Although these workers have mentioned the presence of either or both tissues in trout hepatomas, they have not discussed their importance in controlling the growth or causing the regression of tumors. Hellstrom and Hellstrom ('69) have reviewed the subject of lymphocytic antagonism to tumor cells. The body of literature concerned with host immune reaction in neoplasia is already large and growing rapidly, but apparently there have been no published reports of this phenomenon in trout hepatoma. ANAT. REC., 175: 97-106. 97 98 J. H. WALES AND R. 0. SINNHUBER mate of incidence can be made from the skip-sectioning technique employed. age of such cases must be very small, possibly 10%. OBSERVATIONS Lymphoc ytosis The presence of foci of lymphocytes within a liver normally indicates the presence of disease organisms or injured tissue but in the livers of the trout which have been fed carcinogenic diets the areas of lymphocytosis are usually restricted to hepatic nodules (figs. 9-12). Usually these are nodules which we class as eosinophilic or preneoplastic but occasionally they are assumed to be small hepatomas. Foci of lymphocytes are found more frequently in livers of trout fed a mycotoxin plus cyclopropenoid fatty acids (CPFA) (Sinnhuber et al., ’66). Presumably the CPFA reduces the host defense mechanisms and a greater number of incipient hepatomas reach a recognizable size. These defense mechanisms, we assume, do not include lymphocytosis. The extent to which these small nodules are invaded by lymphocytes is variable but usually it is but a small portion of the entire hepatoma. Frequently we find foci of lymphocytes in areas of the liver sections which are not clearly neoplastic or “preneoplastic” and there may be reasonable doubt about the stimulus which has produced this reaction. In most cases, however, the character of the liver parenchyma being invaded by lymphocytes is that of neoplastic tissue. In most instances the situation is similar to that illustrated in figures 9 and 10 where the distribution of lymphocytes is sharply confined to the abnormal cells. Nearly all of the neoplasms observed in our experimental fish have been trabecular hepatocarcinomas in the various stages of development or evolution which they undergo. The term “hepatoma” will be used in the broad sense to include all the stages which the writers believe are clearly developmental forms or variations of hepatocarcinomas. Possibly the only departure from the common parenchymal cell modification are the pseudo-bile ducts observed in certain hepatomas. These appear to arise within areas of fibrosis and are presumed to be modifications of bile duct epithelium. Fibrosis Our tissue sections indicate that the monocellular strands of loose connective tissue wander from the point of origin, usually near the center of the tumor, toward the periphery of the hepatoma. Apparently the connective tissue cells involved in this process originate from the supporting sheath surrounding blood vessels and bile ducts. These strands become multicellular bundles and eventually form continuous masses of fibrous cells with occasional islets of hepatoma cells enclosed therein (figs. 1-4). As this process continues the center of the tumor becomes almost entirely fibrotic with necrotic spaces containing a semi-fluid mass. The neoplastic parenchyma becomes a shell around the periphery of the fibrotic mass and even this shell of hepatoma tissue may become completely overgrown and presumably ceases to grow (figs. 5-8). This process is highly variable, each tumor may present a slightly different picture. Occasionally the fibrous strands will not terminate their outward growth at the tumor surface but will spread along the surface developing a fibrous sheath over the tumor. If the tumor extends to the surface of the liver it will touch the abdominal wall or some visceral organ such as the pyloric caeca and in such cases the fibrous strands may continue beyond the tumor into the body wall or visceral organ forming adhesions. Very rarely a hepatoma may contain no visible connective tissue hyperplasia but the percent- DISCUSSION Fibrosis Although the term “fibrosis” is applied to the situation which develops within most trout hepatomas it should be made clear that we are referring to both the loose connective tissue cells and the collagenous fibers which accompany them. Microscopically this is quite similar to the aflatoxin-induced “bile-duct proliferation” commonly observed in duckling livers (Asplin and Carnaghan, ’61). However, the two types of hvperplasia seem to be dissimilar etiologically. In the duckling, for example, the proliferation of this connective tissue TROUT HEPATOMA: FIBROSIS AND LYMPHOCYTOSIS 99 occurs in the non-neoplastic hepatic tissue parenchyma interlaced by strands and whereas in the trout this hyperplasia is bundles of connective tissue. We are conrestricted to the hepatoma and only rarely vinced from abundant observations that extends beyond the periphery of the hepa- fibrosis precedes necrosis and we hypothetoma. This difference leads to speculation size that the islets of neoplastic parenabout the stimuli involved. Obviously the chyma, isolated by fibrosis, become neneoplastic tissue is compatible with the crotic. hyperplastic connective tissue and appears Lymphocytosis to stimulate its growth. On the other hand It could be argued that disease organthe normal liver parenchyma appears to be incompatible with the hyperplastic isms or necrosis elicit the lymphocytic reductal cells but compatible with similar sponse but these views lack support. Our duct cells in the normal portion of the fish rarely have diseases of any kind and liver. This resembles the homograft re- no case has yet been found in our laborajection phenomenon and it raises the ques- tory in which a liver had been invaded by tion whether there is a significant degree any organism. In addition we f h d lymof incompatibility between the normal phocytosis in very small, presumably very liver tissue and the hepatoma. The rela- young nodules, less than a millimeter in tionship of normal liver parenchyma to diameter, which are simply colonies of the various elements of the ductal system atypical cells without fibrosis or any newithin the liver indicates that there is com- crosis. Consequently we view these lympatibility but no evidence of stimulation. phoid invasions of the hepatomas as imThis condition appears to be altered when mune reactions to a foreign substance. Our observations lead us to believe that the parenchyma transforms from normal there are two distinct phenomena involved. to neoplastic. Invariably the center of a hepatoma is The connective tissue is “permitted” or the location of the greatest development “stimulated” to hyperplastic growth by the of connective tissue. Apparently a small neoplastic liver parenchyma. The lymphoid colony of hepatoma cells in its early de- tissue, on the other hand, is attracted to velopment will eventually surround a duct. the neoplastic cells, possibly by a substance The connective tissue element of the duct secreted by these malignant cells. We believe that these observations lend is stimulated to hyperplastic proIiferation even though the hepatoma may be a milli- support to our hypothesis that fibrosis and meter in diameter. Normally such hepa- lymphocytosis can, in appropriate circumtomas grow spherically; thus the center will stances, control the growth of, or combe the oldest portion and the area most pletely destroy hepatomas in the rainbow likely to contain connective tissue. We trout liver. believe that the sequence of events is that ACKNOWLEDGEMENT described above rather than the alternative We wish to thank Mrs. L. ,T. Hunter for proposition that necrosis begins in the center of a tumor, due to lack of circula- her assistance in preparing the tissue for tion, and is followed by the connective examination. tissue invasion to occupy the space. We LITERATURE CITED have in our collection of trout hepatomas Ashley, L. M. 1969 Experimental fish neoseveral hundred tumors ranging in diamplasia. In: Fish in Research. Neuhaus and eter from microscopic to 80 mm. Only the Halver, eds. Academic Press, New York-Lonlargest tumors, approximately 40 mm or don, pp. 23-43. 1970 Pathology of fish fed ailatoxins larger, contain areas of necrosis similar to and other antimetabolites. In: A Symposium those shown in figure 13. It is probably true o n Diseases of Fishes and Shellfishes. Snieszko, that connective tissue possibly supports the ed. Special Pub. No. 5, Am. Fish. SOC.,Wash: architecture of the tumor after central ington, D. C., 366-379. necrosis has eroded much of the interior Ashley, L. M., and J. E. Halver 1963 Multiple metastasis of rainbow trout hepatoma. Trans. tumor tissue but prior to this late staqe of Am. Fish. SOC., 92: 365-371. development the tumors are completely Asplin, F.D., and R. B. A. Carnaghan 1961 The filled with vigorously growing neoplastic toxicity of certain groundnut meals for poultry 100 J. H. WALES AND R. 0. SINNHUBER with special reference to their effect on ducklings and chickens. Vet. Record, 73: 1215-1219. Cudkowicz, G., and C. Scolari 1955 Un tomore primitivo epatico a diffusione spizootica della trota iridea di allevamento (Salmo irideus). Tumori, 41: 524-537. Ghittino, P., and F. Ceretto 1962 Studio s d a eziopatogenesi dell' epatoma della trota iridea di allevamento. Tumori, 48: 393-409. Haddow, A., and I. Blake 1933 Neoplasms in fish: a report of six cases with a summary of the literature. J. Path & Bact., 36: 41-47. Hellstrom, K. E.,and I. Hellstrom 1969 Cellular immunity against tumor antigens. Advan. Cancer Res., 12: 167-223. Hueper, W.C., and W. W. Payne 1961 Observations on the occurrence of hepatomas in rainbow trout. J. Nat. Cancer Inst., 27: 1123-1143. Lee, D. J., J. N. Roehm and R. 0. Sinnhuber 1967 Effect of w3 fatty acids on the growth rate of rainbow trout. Salmo gairdneri. J. Nutr., 92: 93-97. Nigrelli, R. F., and S. Jakowska 1961 Fatty degeneration, regenerative hyperplasia and neoplasia in the livers of rainbow trout, Salmo gairdneri. Zoologica, 48: 49-55. Rucker, R. R., W. T. Yasutake and H. Wolf 1961 Trout hepatoma a preliminary report. Prog. Fish-Cult., 23: 3-7. Scarpelli, D. G., M. H. Greider and W. J. Frajola 1963 Observations on hepatic cell hyperplasia, adenoma and hepatoma of rainbow trout (Salmo gabdneri). Cancer Res., 23: 84-57. Sinnhuber, R. O., J. H. Wales, J. L. Ayres, R. H. Engebrecht and D. L. Amend 1968 Dietary factors and hepatoma in rainbow trout (Salmo g a i r d n e r i ) . I. Aflatoxins in vegetable protein feedstuffs. J. Nat. Cancer Inst., 41: 711-718. Sinnhuber, R. O.,J. H. Wales, R. H. Engebrecht, W. D. Kray, et al. 1965 Aflatoxins in Cottonseed meal and hepatoma in rainbow trout. Fed. Proc., 24: 627 (Abstract). Sinnhuber, R. O., J. H. Wales and D. J. Lee 1966 Cyclopropenoids, cocarcinogens for aflatoxininduced hepatoma in trout. Fed. Proc., 25: 555 (Abstract ) . Wales, J. H. 1970 Hepatoma in rainbow trout. In: A S y m p o s i u m on-Diseases of Fishes and Shellfishes. Sniesko, ed. Special Pub. No. 5, Am. Fish. SOC.,Washington, D. C., pp. 351-365. Wood, E. M., and C. P. Larson 1961 Hepatic carcinoma in rainbow trout. Arch. Path., 71: 471-479. - PLATE 1 EXPLANATION O F FIGURES 1 Hepatoma of rainbow trout surrounded by normal liver. The entire hepatoma ( 2 m m diameter) is divided by heavy strands of connective tissue. Hematoxylin and eosin ( H & E). x 20. 2 Hepatoma (4 mm diameter) containing slender strands and masses of connective tissue (arrow). H & E. X 20. 3 Detail of hepatoma (5 m m diameter) showing a cluster of neoylastic parenchymal cells (arrow) surrounded by connective tissue. At this stage of fibrosis there is no evidence of necrosis. H & E. X 128. 4 Central area of hepatoma heavily overgrown by connective tissue. Despite the division and isolation of the hepatoma tissue there is no evidence of necrosis. H & E. X 128. TROUT HEPATOMA : FIBROSIS AND LYMPHOCYTOSIS J. € Wales I. and R. 0.Sinnhuber PLATE 1 101 PLATE 2 EXPLANATION OF FIGURES 5 Details of hepatoma tissue divided by fibrous strands. H & E. X 320. 6 A single isolated group of neoplastic parenchymal cells surrounded by fibrous tissue. H & E. x 800. 7 Detail of hepatoma tissue divided by fibrous strands. H & E. X 320. 8 102 A necrotic islet of hepatoma cells entirely surrounded by connective tissue of bile duct origin. H & E X 320. TROUT HEPATOMA: FIBROSIS AND LYMPHOCYTOSIS J. H. Wales and R. 0.Sinnhuber PLATE 2 PLATE 3 EXPLANATION O F FIGURES 9 Section of trout liver containing a small hepatoma approximately 1 mm diameter. Note that within the hepatoma are several darkly staining foci of lymphocytes. H & E. x 20. 10 Margin of darkly staining hepatoma with several areas occupied by lymphoid tissue. H & E. x 128. 11 Central portion of hepatoma heavily invaded by lymphoid tissue. H & E. X 128. 12 Detail of hepatoma which has been occupied by lymphoid tissue leaving a few isolated clusters of neoplastic parenchyma. H & E. x 320. 104 TROUT HEPATOMA: FIBROSIS AND LYMPHOCYTOSIS J. H. Wales and R. 0. Sinnhuber PLATE 3 105 TROUT HEPATOMA: FIBROSIS AND LYMPHOCYTOSIS J. H. Wales and R. 0.Sinnhuber PLATE 4 EXPLANATION OF FIGURE 13 Longitudinal section through the liver of a two year old rainbow trout. Note the single, large tumor composed largely of neoplastic parenchyma. The center of this tumor contains connective tissue and vacuoles of necrotic hepatocytes. Another smaller tumor (above) has been completely overgrown by connective tissue and its growth has presumably ceased. A fragment of normal liver (left) remains. x 2.5. 106
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